Apparatus for measuring spatial distribution of fluorescence on a

Abstract

A fluorescence detection scanner and method employing a fiberoptic collector, positioned adjacent to the scanning plane of the excitation beam, with a light collecting surface which is oriented to reject back-scattered excitation light from the incident surface of the sample support. The scanning plane of the excitation beam is off normal relative to the incident surface of the sample support and the light collecting surface is located within the area defined by the resulting acute angle of incidence. The light collecting surface is angled away from the location at which the incident excitation beam intersects the surface of the sample support so that back-scattered excitation light does not enter the optical fibers. The orientation of the light collector results in a four to five-fold decrease in excitation-light background without attenuation of the fluorescence emitted by the sample. Long-pass interference filters, selected to reject the excitation wavelength, are located at the input and output surfaces of the fiberoptic light collector to further reduce background excitation light. Multiple fluorophores are discriminated by sequentially scanning the sample with a different interchangeable narrow bandpass filter for each fluorophore. Internal fluorescence standards in the sample are used to determine filter efficiencies for each label in each filter image and the quantity of each fluorophore is computed by linear analysis. Increased detector sensitivity allows linear quantitation of multiple fluorophores in the femto-mole range.